FR2800077A1 - Chimeric transcription factor, useful for detecting specific binding interactions between proteins, is fusion of Pol III transcription factor and DNA-binding domains and includes a conditional mutation - Google Patents

Abstract

Chimeric transcription factor (I) comprises a fusion protein of domain I (A), i.e. a subunit of a Pol III transcription factor, and domain II (B), i.e. a DNA-binding peptide domain, and where at least one domain is a conditional mutant. Independent claims are also included for the following: (1) nucleic acid (II that encodes (I); (2) recombinant vector comprising (II); (3) yeast cell (III) transformed with (II); (4) method (M1) for detecting interactions between two peptides, or peptide domains; (5) kit for performing (M); and (6) method M2) for selecting interaction partners with a selected peptide (or domain).

Description

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CHIMERIC TRANSCRIPTION FACTOR ACTIVITY
CONDITIONAL, AND ITS USES FOR DETECTION
INTERACTIONS BETWEEN PROTEINS.

 The present invention relates to novel methods for detecting and analyzing interactions between proteins or peptide sequences.

 The study of interactions between proteins or sub-domains of proteins is an essential approach for the understanding of fundamental cellular mechanisms, integrated or isolated molecular mechanisms or for the screening of molecules with biological actions, etc.

 The so-called "double-hybrid method" for detecting protein-protein interactions has been developed by FIELDS & SONG (FIELDS & Song, Nature 340,245-246, 1989, US Patent 5283173).

This method is based on the co-expression, in a yeast cell: of a reporter gene, whose open reading frame is placed under transcriptional control of a promoter regulated by regions allowing the attachment of the binding domain to DNA (called BD domain, for Binding Domain) of an activator of transcription of RNA polymerase
II (PolII); a chimeric gene coding for a fusion protein between a first test protein and a BD domain capable of binding on the regions of the DNA controlling the reporter gene; a chimeric gene coding for a fusion protein between a second test protein and an activation domain (AD for Activating
Domain) of a PolII transcription activator.

 If both fusion proteins interact, a functional transcription activator is

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 reconstituted, and the expression of the reporter gene is observed.

 This system makes it possible to study the interactions between two known proteins (or peptide domains) or to search, by screening fusion libraries, for proteins that can interact with a given protein.

 Initially, the double-hybrid system was developed in S. cerevisiae, placing the reporter gene under the UASG (Upstream Activating Sequences of GAL genes) sequences. These sequences are recognized by the Gal4p transcription activator. In this case, one of the chimeric genes encodes a protein fused to the Gal4p BD domain (amino acids 1-147) while the other gene encodes a protein fused to the Gal4p AD domain (amino acids 769-881). Many variations of the dual-hybrid system have been developed. However, the use of reporter genes transcribed by RNA polymerase II limits the applications of this system. In particular, this technique does not make it possible, for example, to study interactions involving a protein capable of activating transcription by RNA polymerase II.

 To overcome this limitation, a system hereinafter referred to as double-hybrid PolIII has been developed; this system is described in particular in the PCT Application WO / 96/37618 in the name of the Atomic Energy Commission, as well as in the publication of PCT Application WO / 96/376181, or in the publication of MARSOLIER and SENTENAC, [Methods in Enzymology, 303, 411-422 (1999)].

 The double-hybrid PolIII system is based on the use, as a reporter gene, of a gene transcribed by RNA polymerase III. In this reporter gene, a region allowing the attachment of a transcription factor PolIII is inactivated and replaced by a region allowing the attachment of a

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 BD domain of a PolII transcription activator. This reporter gene is expressed in the same cell as a chimeric gene coding for a fusion protein combining one of the test proteins with the BD domain of said transcriptional activator PolII, and a chimeric gene coding for a fusion protein. associating another test protein with a subunit of said PolIII transcription factor. In case of interaction between the 2 proteins, a functional transcription factor is reconstituted, and the expression of the reporter gene is observed.

 This system has for example been implemented using the SNR6 gene as a reporter gene; this gene, which is transcribed into U6 RNA involved in splicing introns, is essential for cell survival. A region of the SNR6 gene called box B (which allows the attachment of the T138 subunit of the transcription factor PolIII TFIIIC, and is necessary for the transcription of this gene), is inactivated and replaced by UASG sequences. This reporter gene (called UASG-SNR6) is associated with 2 chimeric genes; the first code for a fusion protein associating one of the test proteins with the Gal4p BD domain (amino acids 1-147); second code for a fusion protein associating the other test protein with the subunit # 138 of the transcription enhancer PolIII TFIIIC.

 When these constructs are introduced into the same yeast strain, the UASG-SNR6 gene is transcribed only if there is an interaction between the 2 test proteins.

 This transcription can be detected by quantification of transcripts, or by a cell survival test.

 In the first case, the reporter gene UASGSNR6 is modified (for example by inserting an oligonucleotide in the transcribed portion) so as to

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 its transcripts can be distinguished from those of the wild-type SNR6 gene present in the cell.

In the second case, a yeast strain whose chromosomal SNR6 gene is inactivated is used, and the survival is ensured by the presence of a plasmid carrying the wild-type SNR6 gene, and the selection marker URA3. Due to the presence of this marker, this strain does not grow in the presence of 5-fluoro-orotic acid (5FOA). When the 3 constructions mentioned above are introduced into this strain cultivated in the presence of 5-FOA, it can only grow if it loses the plasmid bearing the URA3 marker and the wild-type SNR6 gene, that is to say there is an interaction between the 2 test proteins, allowing the transcription of the UASG-SNR6 gene
To further facilitate the use of the double-hybrid PolIII system, particularly in the context of the screening of active molecules, the inventors have now developed a new system for conditional selection of interactions.

 This system is based on the use of transformed yeast cells, in which the wild-type copy (s) of a gene X transcribed by polymerase III, and providing a vital function for the cell, have ) been inactivated (s). In replacement of the X gene, these cells comprise an X 'gene capable of performing the same vital function as the X gene, and derived from this gene by the inactivation of a region (hereinafter referred to as SC region) of the promoter. PolIII of said gene, specifically recognized by a transcription factor PolIII and allowing the attachment of said transcription factor, and its replacement by a region (hereinafter referred to as SC 'region) allowing the attachment of another peptide domain binding to the DNA. In the same cell is expressed a chimeric transcription factor PolIII, whose field of

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 DNA binding is able to bind specifically to the SC 'region, and whose ability to activate transcription by PolIII is conditional. The vital function can only be ensured through the transcription of the X 'gene, which can only be performed under permissive conditions for the activity of said chimeric transcription factor.

 These cells can be transformed by vectors of a double-hybrid PolIII system expressing proteins whose interaction it is desired to test. These vectors are chosen so that, if there is an interaction between the 2 proteins tested, the vital function can be ensured and the cells are viable under non-permissive conditions for chimeric transcription factor activity.

 The subject of the present invention is a chimeric transcription factor consisting of a fusion protein comprising: a domain I chosen from: the subunits of a transcription factor PolIII, and the conditional mutants of said subunits, and a Domain II selected from: DNA binding peptide domains and conditional mutants of said domains; characterized in that at least one of domains I or II is a conditional mutant.

 A mutation is defined as a conditional mutation that manifests itself as a wild-type phenotype under certain conditions, called permissive conditions, and by a mutant phenotype under other conditions, called non-permissive conditions or restrictive conditions.

 A conditional mutant of a subunit of a transcription factor PolIII is a mutant of one of the subunits of a factor TFIIIB or TFIIIC capable of

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 participate in the activation of transcription by PolII RNA, under permissive conditions, but not under restrictive conditions.

 A DNA binding peptide domain is a peptide capable of specifically recognizing a DNA target sequence, thereby enabling the attachment at said target sequence of any protein comprising said peptide.

 A conditional mutant of a DNA binding peptide domain is a mutant capable of binding, under permissive conditions, but not under restrictive conditions, to the target sequence recognized by said domain.

 To obtain a chimeric transcription factor according to the invention, the domain I will preferably be selected from the group consisting of the subunits # 138, # 131, i95 and r91 of the TFIIIC factor, and the conditional mutants. said subunits.

 Advantageously, the domain I consists of a thermosensitive mutant of # 138. For example, domain I consists of a mutant of # 138 obtained by deletion of all or part of the central domain (amino acids 330-760), and which comprises at least the Cterminal domain (amino acids 761-1160) of # 138 and preferably also includes the N-terminal domain (amino acids 1-329) of # 138.

 The inventors have indeed found that certain # 138 mutations, and in particular deletions of all or part of the central domain (amino acids 330-760), which did not seem to affect the activation of the transcription of UASG-SNR6 when they are fused to Gall-147 [MARSOLIER et al., 1997, J. Mol. Biol. 268, 243- 249], are heat-sensitive mutations, the effect of which is manifested from 37 C until complete inhibition of transcriptional activation at 38 C.

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 For the purpose of obtaining a chimeric transcription factor according to the invention, domain II will be chosen from DNA binding domains other than that of the subunit of transcription factor PolIII of domain I. non-limiting examples, it may be chosen in particular from the DNA binding domains of transcription activators PolII, such as the 1-147 domain of the GAL4 transcription activator, which specifically recognizes the UASG sequences; the LexA binding domain [BRENT and PTASHNE, Cell, 43, 729-736, (1985)] or that of nuclear hormone receptors such as estrogens [LE DOUARIN et al. Nucleic Acids Research, 23, 876-878, (1995)], glucocorticoids, growth hormone, and conditional mutants of said DNA binding domains; it may also be selected from Rapl DNA binding domains [GILSON et al. J. Mol. Biol., 231, 233-310 (1993)], ORC6, [LI and HERSKOWITZ, Science, 262, 1870-1874 (1993)], and their conditional mutants.

 According to a preferred embodiment of the present invention, domain II is a thermosensitive mutant of the DNA binding domain of GAL4 (Gal4BD). The inventors have, for example, shown that the P26L mutation of Gal4BD (replacement of proline at position 26 by a leucine), whose previously known effect was a modification of the capacity of Gal4p to bind to DNA as a function of Zn2 + ion concentration [JOHNSTON Nature, 328, 353-355, (1987); JOHNSTON and DOVER, Genetics, 120, 63-74, (1988)], was also a partially thermosensitive mutation, the effect of which manifests itself from 37 C.

 The subject of the present invention is also nucleic acid sequences coding for chimeric transcription factors in accordance with the invention, as well as recombinant vectors, in particular vectors.

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 expression system comprising said sequences, and cells, in particular yeast cells, transformed by said sequences and expressing a chimeric transcription factor according to the invention.

 Yeasts-hosts used for the implementation of the present invention are in particular yeasts of the genus Saccharomyces, in particular of the species Saccharomyces cerevisiae.

 However, it is also possible to use yeast cells belonging to other genera, for example Schizosaccharomyces pombe cells.

 Expression vectors in accordance with the invention comprise at least one nucleic acid sequence coding for a chimeric transcription factor according to the invention, associated with active transcriptional control elements in yeast. Vectors, as well as transcription control elements, usable in yeast and allowing the construction of expression vectors according to the invention are known per se, and will be chosen in particular according to the yeast-host that we want to use.

 When domain II is a thermosensitive mutant of the GAL4 DNA binding domain, a nucleic acid sequence according to the invention will preferably be placed under transcriptional control of a weak promoter. As an example of a weak promoter that can be used in Saccharomyces cerevisiae, the promoter CYC1 carried by the vector p416CYC1 [ATCC 87384; Mummberg et al., Gene, 156, p192 (1995)].

 Expression vectors in accordance with the invention also advantageously comprise a selection marker, for example a URA3 marker allowing their removal (for example by a 5-FOA flush in the case of a URA3 marker). This allows the

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 if necessary, eliminate the chimeric activator to confirm that it is not functional under restrictive conditions.

 Transformed cells according to the invention are preferably cells in which an X gene, normally transcribed by RNA polymerase III and indispensable for cell viability, has been inactivated, and which comprise at least one copy of an X 'gene. derivative of said gene X by inactivation of a promoter SC region of said gene for the attachment of a transcription factor by polymerase III and insertion of a SC 'region permissive for the fixation of a transcription factor chimeric transcription according to the invention.

 Advantageously, said cell further comprises a gene X '' derived from the X gene by inactivation of the SC region, and its replacement by a SC '' region, different from SC ', and allowing the attachment of a peptide binding domain to DNA other than domain II of a chimeric transcription factor according to the invention.

 By way of nonlimiting examples: if the X gene is the SNR6 gene, the SC region is preferably the B block; In this case, the X 'gene and, if appropriate, the X' 'gene will both be derived from SNR6 by inactivation of the B block, and addition of a recognized SC region under permissive conditions, respectively, by the domain II. a chimeric transcription factor according to the invention, and a SC '' region recognized by a DNA binding peptide domain other than said domain II.

 if the domain II of a chimeric transcription factor according to the invention is a conditional mutant of the BD domain of GAL4, the SC 'region of the X' gene will comprise one or more UASG sequence (s) in this case, the region 'SC' of the gene X '' will be able to

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 example include one or more operator (s) LexA, or one or more element (s) of response to a nuclear hormone receptor; if the domain II of a chimeric transcription factor according to the invention is a conditional mutant of the LexA BD domain, the SC 'region of the X' gene will comprise one or more LexA operator (s), and the SC '' region. the X "gene may for example comprise one or more UASG sequence (s), or one or more element (s) for response to a nuclear hormone receptor.

 The X 'gene and / or the X' 'gene may be integrated into the chromosome of the cell, or carried by extra-chromosomal DNA, for example a plasmid. Preferably, at least one copy of the X 'gene is integrated into the chromosome of the cell, for example at the location of the wild-type X gene. Additional copies may be carried by plasmids.

 The transcription of the X 'gene in cells according to the invention as described above can only be carried out via a conditional activity chimeric transcription factor according to the invention, and under permissive conditions. for the activity thereof, and the X '' gene is not transcribed.

 Transformed cells according to the invention can be used as host cells in the framework of the conditional selection system for the protein interactions mentioned above.

 The subject of the present invention is a method for detecting the interactions between two peptides or two peptide domains Ti and T2, which process is characterized in that it comprises: i) the co-transformation of at least one yeast cell such that defined above, expressing a chimeric transcription factor according to the invention, and in which an essential X gene has

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has been replaced by an X 'gene comprising a region
SC 'for fixing said chimeric transcription factor, by the following DNA sequences: a DNA sequence (a) comprising a sequence coding for a fusion protein comprising the peptide or the peptide domain Ti, and a subunit unit of a PolIII transcription factor; a DNA sequence (b) comprising a sequence coding for a fusion protein comprising a DNA binding peptide domain capable of binding to the SC 'region of the X' gene, and the T2 peptide or peptide domain ; ii) determining the viability of the cell placed under non-permissive conditions for the chimeric transcription factor expressed by said host cell.

 Prior to their introduction into a cell according to the invention, the sequences (a) and (b) were respectively placed in appropriate vectors allowing their expression in said cell. Useful vectors for the expression of sequences (a) and (b) are for example those, known in themselves, which allow the expression of a gene in a yeast cell, and in particular those which are used in the framework for the implementation of the double hybrid methods PolII and double-hybrid PolIII known in the prior art.

 The subunit of a transcription factor PolIII (a), as well as the DNA binding peptide domain encoded by sequence (b) are, of course, functional under permissive or non-permissive conditions for the transcription factor. chimeric transcription expressed by the host cell according to the invention.

 During the implementation of the method according to the invention, when the cell is placed in conditions

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 non-permissive for the chimeric transcription factor, transcription of the X 'gene can not take place, and the cell can survive, only if there is interaction between the T1 and T2 proteins, via the double hybrid transcription constituted by this interaction.

 According to a variant of the process according to the invention, the host cell further comprises a gene X '' as defined above: in this case, the sequence (b) comprises instead of the sequence encoding a peptide binding domain. DNA capable of binding to the SC 'region of the X' gene, a sequence encoding a DNA binding peptide domain capable of binding to the SC '' region of the X gene.

 When this variant is implemented, when the cell is placed in non-permissive conditions for the chimeric transcription factor according to the invention, transcription of the X 'gene can not take place. The cell can only survive if the transcription of the X '' gene can be effected via the double hybrid transcription factor constituted by an interaction between the T1 and T2 proteins.

 According to a preferred embodiment of the present invention, said chimeric transcription factor is heat-sensitive, and the cell is placed, in step ii) at a temperature greater than or equal to 37 C, preferably at a temperature of the order from 38 C.

 According to a preferred embodiment of the method of the present invention, when the chimeric transcription factor expressed by the host cell is encoded by a vector carrying a selection marker allowing its elimination, said method may comprise, if appropriate, a additional step of removing said vector, which avoids possible falsepositifs.

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 For example, if the chimeric transcription factor expressed by the host cell is encoded by a plasmid carrying a URA3 marker, the viable cells selected at the end of step ii) of a process according to the invention are postponed. in culture in the presence of 5-FOA. Under these conditions, the only cells capable of growing are those which can lose the plasmid bearing the URA3 marker and the coding sequence for the chimeric transcription factor, that is to say those in which the transcription of the X 'gene is carried out by the intermediate of the interaction between T1 and T2 proteins.

 The present invention is particularly suitable for the screening of libraries, making it possible to search for potential partners for interaction with a given protein or protein domain. Such libraries are, for example, fusion libraries with a transcription activator, consisting of a plurality of different sequences randomly fused with the same transcription activation protein domain.

 For example, for the search for potential partners of interaction with a polypeptide or peptide domain T, the process according to the invention comprises: i) the co-transformation of at least one yeast cell according to the invention by a DNA sequence (a) comprising a sequence coding for a fusion protein comprising the polypeptide or the T peptide domain and a DNA binding peptide domain capable of binding on the SC 'region of the X' gene or a DNA binding peptide domain capable of binding to the SC '' region of the X '' gene; and a DNA sequence (b) randomly taken from a bank of test sequences, each of which codes for a fusion protein comprising a subtype.

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 a unit of a PolIII transcription factor and a Tx peptide polypeptide or domain candidate for interaction with the T protein, and; ii) selecting viable transformed cells under non-permissive conditions for the chimeric transcription factor according to the invention expressed by said cell; iii) sequencing the DNA sequence (b) recovered from the viable cells, and determining the sequence of the polypeptide or Tx peptide domain.

 Alternatively, the method according to the invention can also be used to screen a library of test sequences fused to a DNA binding peptide domain capable of binding to the SC 'region of the X' gene, or well with a DNA binding peptide domain capable of binding to the SC '' region of the X '' gene, using a T sequence fused to a subunit of a PolIII transcription factor.

 The present invention also relates to kits for implementing the method according to the invention.

 Such kits comprise, for example: at least one yeast strain according to the invention, associated with: at least one vector comprising a DNA sequence coding for a subunit of a transcription factor PolIII and at least one a site for insertion of a sequence encoding a polypeptide fused to said subunit, and / or - at least one vector comprising a DNA sequence encoding a fusion protein comprising a peptide binding domain DNA capable of binding to the SC region of an X 'gene, and at least one site allowing the insertion of a coding sequence for a polypeptide fused to said domain, or

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 at least one vector comprising a DNA sequence coding for a fusion protein comprising a DNA binding peptide domain capable of binding to the SC region of an X '' gene, and at least one site allowing inserting a coding sequence for a fused polypeptide into said domain.

 Advantageously, kits in accordance with the invention also comprise control vectors, in particular positive interaction control vectors.

It may for example be a pair of vectors, one of which comprises a DNA sequence coding for the BD domain of a transcriptional activator PolII, and the other comprises a DNA sequence coding for a subunit of a transcription factor PolIII, respectively fused to sequences encoding proteins known to interact with each other.

 The present invention will be better understood with the aid of the additional description which follows, which refers to an example of obtaining and implementing a conditional system for detecting interactions between proteins according to the invention.

EXAMPLE: I-VECTORS AND STRAINS:
The vectors described below were constructed by selecting auxotrophy markers compatible with the double hybrid system PolII.

Notably, the majority of existing dual-hybrid PolII systems generally use TRP1-tagged plasmids for the expression of Gal4BD-fused proteins (1-147), and LEU2-labeled plasmids for the expression of fused proteins at the same time. transcription activation domain.

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Vector allowing the expression of proteins fused to the Gal4p DNA binding domain: pQBD1
A vector allowing the expression of proteins fused to the Gal4p DNA binding domain (Gal4BD) was made using a cloning multisite compatible with the most widely used double-hybrid PolII vectors.

 The vector pODB8 (LOUVET O. et al., 1997, BioTechniques 23 (5), 816-820, GENBANK N U57443) was digested with the restriction endonuclease NheI. The single-stranded ends generated by this enzyme were digested by MUNG BEAN NUCLEASE to obtain blunt-ended ends. The vector was then digested with the enzyme EcoRI. The cohesive end generated by EcoRI was transformed into a free edge by the action of the Klenow fragment. The vector was then religated on itself. The vector is named pQBD1 (Figure 1).

 An oligonucleotide hybridizing upstream of the cloning multisite of pQBD1 was designed and tested to allow verification by DNA sequencing of the clonings performed.

 The sequence of this oligonucleotide, called qbdmcs is: ATCATCGGAAGAGAGTAG.

 The cloning multisite of the pQBD1 and its reading phase are compatible with those of the conventional double-hybrid PolII pGBT9 vectors [BARTEL et al., HARTLEY Ed. Oxford University Press, 153-179, (1993)] pAS1, pAS2, pAS2- [HARPER et al., Cell, 75, 805-816, (1993)] pODB8, pODB80 [LOUVET et al., BioTechniques, 23, 816-820, (1997). This makes it easy to transpose the DNA inserts of these vectors to pQBD1. In addition, the auxotrophy marker of this vector (TRP1) is the same as that used for vectors of the same type in the double-hybrid system PolII.

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 Thus, in a single cloning, a protein can be tested in a PolII double-hybrid system, and transferred into a conventional dual hybrid PolIII system, or in a conditional system according to the invention.

Positive control expressing the Gal4BD-Tl38 fusion protein
In plasmid pQBD1, the coding sequence for the T138 protein was fused with the coding sequence for Gal4BD. This fusion protein allows activation of the transcription of the UASG-SNR6 reporter gene. This vector was designed to serve as a positive control for the PolIII double hybrid system.

 For this, the vector pOL98 (resulting from the insertion of # 138 flanked by BamHI sites in pBLUESCRIPT) was digested with the PvuI enzyme and then the BamHI enzyme. The 3494 bp BamHI-BamHI fragment corresponding to the # 138 open reading frame was inserted into the pQBD1 vector at the BamHI site. The integration direction of the insert was verified by enzymatic digestion. The vector is named pQBD / tl38 (Figure 2).

Vector allowing the expression of proteins fused to the protein # 138 of the TFIIIC complex
A vector allowing the expression of the proteins fused to the protein # 138 of the TFIIIC complex was constructed. The open reading frame encoding # 138 was placed in the multicopy vector p423GPD (MUMBERG et al., 1995, Gene 156,119-122; ATCC N 87355) under the control of the strong S. cerevisiae GPD promoter. This vector is selectable thanks to the HIS3 marker.

 For its production, the pGEN / T138 vector (MARSOLIER et al., J. Mol Biol., 1997, publication cited above) was digested with the enzyme BamHI. The 3492 bp fragment corresponding to the T138 open reading frame was inserted at the BamHI site of p423GPD. The direction of insertion

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 # 138 in the vector has been verified. The 5G BamHI site at # 138 ATG was deleted to release an additional site in the cloning multisite (MCS) of the vector. For this the vector obtained was digested in a controlled manner by the enzyme BamHI (partial digestion: site on 2). The product of the partial digestion has undergone the action of the Klenow fragment in order to generate blunt ends. The vector was then religated on itself. The disappearance of the BamHI site upstream of # 138 has been verified. This vector has been named pTAUl.

 Three STOP codons were introduced in the different reading frames at the MCS to allow the translation of the fusion proteins to stop. In addition, a new unique cloning site (NotI) was introduced into the MCS and the EcoRI site (non-unique) was removed. For this, the pTAU1 was digested with the enzymes BamHI and XhoI and the MCS was removed.

 This vector was then religated in the presence of a mixture of the oligonucleotides linker-3: TCGATTACTTACTTACTCGAGGTCGACGATATCTGGCGGCCGCCCGGGG and linker + 5: GATCCCCCGGGCGGCCGCCAGATATCGTCGACCTCGAGTAAGTAAGTAA hybridized with each other.

 The resulting vector is named pTAU2 (Figure 3).

The oligonucleotide taumcs.

AATGGCTCCTAGAAAGAC makes it possible to sequence the inserts cloned in the pTAU2 vector.

Yeast strain where the chromosomal SNR6 gene is replaced by the reporter gene UASG-SNR6 .:
A yeast strain that can be used for the implementation of the present invention was obtained from the strain of Saccharomyces cerevisiae W303-1A [MATa, ura3-1, leu2-3,112, trp1-1, his3-11,15, ade2-1, canl 100].

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 In strain W303-1A, the chromosomal SNR6 gene has been replaced by the UASG-SNR6 reporter gene. This was done in order to avoid reversals of the SNR6 :: AB-box locus contained in the initial strain (yMCM616). For this purpose, an integrative vector has been constructed: it carries the selection marker URA3 and allows insertion of the UASG-SNR6 cassette at the SNR6 locus.

The pRS315 / UASG-SNR6 vector (MARSOLIER et al., 1997, JMB, 268, 243-249) was digested with the restriction endonucleases SstI and KpnI. The 662 bp fragment containing the UASG-SNR6 cassette (579 bp EcoRI-BamHI fragment) was inserted into the pRS306 vector cloning multisite (SIKORSKI and HIETER, 1989, Genetics 122 (1), 19-27; U03438) replacing the SstI-KpnI fragment.

The resulting vector is named pRS306 / UASG-SNR6.

 The vector pRS306 / UASG-SNR6 was digested with the enzyme EcoNI (single site within SNR6). This plasmid, linearized, was used for the transformation of the W303-1A strain. The transformants were selected on medium devoid of uracil.

 The integration of the pRS306 / UASG-SNR6 vector at the SNR6 locus was verified by PCR.

 The strain W303-1A + pRS306 / UASG-SNR6 was then transformed with the plasmid pAS2 / # 138 expressing the Gal4BD-# 138 fusion, and with the plasmid pRS315 / SNR6-UASG (M.C.

Marsolier, J. Mol. Biol., 1997, publication cited above).

Transformants were selected on media lacking tryptophan and leucine. Strain W303lA + pRS306 / UASG-SNR6 + pAS2 / # 138 + pRS315 / UASG-SNR6 was placed on medium containing 5-FOA in order to select the excision rearrangement of pRS306 / UASG-SNR6.

Removal of the wild-type copy of the SNR6 gene is verified by PCR on the 5-FOA resistant clones. A recombinant strain which has lost the wild-type SNR6 gene has thus been isolated. This strain W303 / UASG-SNR6 + pAS2 / # 138 + pRS315 / UASG-SNR6, which contains only the reporter gene

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 UASG-SNR6, has the genotype: [MATa, ura3-1, leu2-3,112, trp1-1, his3-11,15, ade2-1, can1-100, SNR6ABbox :: UASGx5].

Vector expressing the UASG-SNR6 reporter gene
The strain W303 / UASG-SNR6 + pAS2 / # 138 + pRS315 / UASG-SNR6 was transformed with the vector pRS316 / SNR6 (centromeric vector, URA3, carrying the wild-type SNR6 gene, MARSOLIER MC, 1997, publication cited above). The strain W303 / UASG-SNR6 + pAS2 / # 138 + pRS315 / UASG-SNR6 + pRS316 / SNR6 was then cultured on a medium containing tryptophan and leucine in order to allow the loss of the various vectors. This made it possible to isolate a strain W303 / UASG-SNR6 + pRS316 / SNR6 having lost the vectors pAS2 / # 138 and pRS315 / UASG-SNR6.

 A multicopy vector (2) carrying the UASG-SNR6 reporter gene and the ADE2 selection marker (very little used by the other yeast vectors) was constructed. The 662 bp SstI-KpnI fragment of pRS315 / UASG-SNR6 containing the UASG-SNR6 cassette was inserted into the pASZ12 vector (STOTZ and LINDER P., 1990, Gene 95, p91-98) replacing the SstI-KpnI fragment. .

The vector has been named pQARl. This plasmid was introduced into the strain W303 / UASG-SNR6 + pRS316 / SNR6. The strain W303 / UASG-SNR6 + pRS316 / SNR6 + pQAR1 has been named QYN1.

 The inventors have shown that the sensitivity of the reporter system is increased in this strain containing a multicopy vector.

 The strain W303 / UASG-SNR6 + pRS316 / SNR6, as well as the QYN1 strain can be used in a conventional double-hybrid PolIII system. To use them in a conditional system according to the invention, they are previously transformed with pQBD / # 138, which makes it possible to eliminate the plasmid pRS316 / SNR6.

Positive interaction control vectors
Two vectors have been constructed to serve as positive controls of interaction. A couple of

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 Prp9p and Prp21p proteins, capable of interacting, were chosen, and the corresponding genes were cloned into expression vectors designed for this purpose.

Vector expressing the Gal4BD-Prp9 fusion protein
Construction of the vector expressing the Gal4BD-Prp9 fusion protein was performed in pQBD1. The vector pPL164 (PRP9 in the plasmid pMA424) was digested with the enzyme SalI and then with the enzyme SmaI. The 1.7 kbp fragment corresponding to PRP9 was inserted into the vector pQBD1 instead of the SmaISalI fragment. The resulting vector was named pQBD / PRP9 (Figure 4).

Vector expressing fusion protein # 138-Prp21
Construction of the vector expressing the fusion protein # 138-Prp21 was undertaken in the pTAU1 vector. The vector pPL247 (PRP21 in pBLUESCRIPT SK) was digested with the enzymes BamHI and SalI. The 873 bp fragment corresponding to the open frame of PRP21 was inserted into the pTAU1 vector replacing the BamHI-SalI fragment. In order to phase the PRP21 open reading frame with that of # 138, the resulting vector was digested with the BamHI enzyme. The generated cohesive ends have undergone the action of the Klenow fragment in order to obtain sharp ends. The vector was then religated on itself. The loss of the BamHI site allows the phasing of the two open reading frames. The resulting vector is named pTAU / PRP21 (Figure 5).

II-THERMOSENSIBLE SELECTION SYSTEM: Vector allowing the expression of a thermosensitive mutant of Gal4BD fused to the protein # 138
The sequence coding for a fusion protein between the P26L mutant of Gal4BD and T138 was carried out and then cloned on the p416CYCl vector. The CYC1 promoter of the centromeric vector p416CYCl allows a quantitatively weak expression of the genes it controls. This

<Desc / Clms Page number 22>

 vector was chosen because the conditional binding of Gal4P26L to DNA was observed when the expression of the corresponding gene is controlled by its own (low level) promoter.

 For this construct, two PCRs were performed on GAL4BD in order to introduce the P26L mutation by changing 2 codon nucleotides to avoid reversions (Proline CCG codon changed to Leucine CTT codon).

The first PCR amplification was performed using the following oligonucleotides: dbxba: CTCCTTCTAGATGAAGCTACTGTCTTC dblfa: GCGCACTTAAGTTTTTCTTTGGAGCAC
The dbxba oligonucleotide hybridizes at the level of the GAL4BD ATG and introduces an XbaI site upstream of the initiation codon. The oligonucleotide dblfa hybridizes on GAL4BD at codon 26 and allows the change of the two nucleotides. In addition, he introduces an AflII site.

The second PCR amplification was carried out using the following oligonucleotides: dbafl: GAAAAACTTAAGTGCGCCAAGTGTCTG dbbam: GAAAAACTTAAGTGCGCCAAGTGTCTG
The oligonucleotide dbafl hybridizes on GAL4BD at codon 26 and allows the change of the 2 nucleotides. In addition, he introduces an AflII site.

 The dbbam oligonucleotide hybridizes at codon 147 of GAL4BD and introduces a BamHI site thereafter.

 The 97 bp fragment generated by the first PCR was digested with XbaI and Enzyme enzymes. The 383 bp fragment generated by the second PCR was digested

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 by AflII and BamHI enzymes. These two PCR products were inserted into the pBLUESCRIPT KS vector in replacement of the XbaI-BamHI fragment. The resulting vector was named pKS / GAL4BDP26L.

 The vector pKS / GAL4BDP26L was digested with the enzymes XbaI-BamHI and the 463 bp fragment corresponding to GAL4BDP26L was inserted into the vector p416CYCl previously purified on gel after XbaI and BamHI digestion. The resulting vector was named p416CYCl / GAL4BDP26L.

p416CYCl/GAL4BDP26L-i138. The vector pQBD / # 138 was digested with the enzyme BamHI. The 3492 bp fragment corresponding to # 138 was inserted into p416CYCl / GAL4BDP26L previously digested with BamHI. The integration direction of the fragment has been verified and the resulting vector has been named

p416CYCl / GAL4BDP26L-I138.

The p416CYCl / GAL4BDP26L-Tl38 vector was introduced into the strain W303 / UASG-SNR6 + pQAR1 + pQBD / # 138 (obtained from QYN1 by elimination of pRS316 / SNR6). After removing the plasmid pQBD / Tl38, the thermosensitivity of this strain was tested.

 For this purpose, the cells containing the vector p416CYC1 / GAL4BDP26L-T138, or, as a positive control, the vector pQBD / # 138, were cultured overnight in a selective medium, diluted to an OD of 0.2 in the same medium and put to push 8 extra hours.

The cultures were diluted 10,000 fold and 100 μl dilutions were plated and incubated at 30 ° C or 38 ° C. The petri dishes were examined after 4 days.

 The results are illustrated in Table I.

l,;onstructlons Nomore ce colonies a 3U"c Nombre de colonies a 3t"C; GAL4(1-147) " -i138 100 15 GAL4-(1-147)-'t138 110 70 La soucne contenant le vecteur

p4l6CYCl/GAL4BDP26L-zl38 est capable de pousser à 30 C mais pousse beaucoup moins bien à 38 C que la souche Table I

Constructors Nominate this colony at 3 ° C Number of colonies at 3 ° C; GAL4 (1-147) "-1388 100 GAL4- (1-147) - '138 110 70 The skyer containing the vector

p4l6CYCl / GAL4BDP26L-zl38 is able to grow at 30 C but grows much worse at 38 C than the strain

<Desc / Clms Page number 24>

 witness. This demonstrates the thermosensitive nature of the Gal4BDP26L mutation.

vecteur p4l6CYCl/GAL4BDP26L-Tl38, la croissance normale de cette souche est rétablie à 38 C. En outre, elle pousse alors sur milieu contenant de l'uracile et du 5-FOA. In addition, when plasmids pQBD / PRP9 and pTAU / PRP21 are introduced into the strain containing the

p4l6CYCl / GAL4BDP26L-Tl38 vector, the normal growth of this strain is restored to 38 C. In addition, it then grows on medium containing uracil and 5-FOA.

Vectors allowing expression of a # 138 thermosensitive mutant fused to Gal4BD protein
Previous works (MARSOLIER et al., J.

Mol. Biol., 1997, publication cited above) have made it possible to highlight domains of # 138 whose fusion with Gal4 (1-147) allowed the activation of the UASG-SNR6 reporter gene. In particular, the two fusions, # 3- Gal4 (1-147) and # 1-3-Gal4 (1-147), respectively comprising amino acids 761 to 1160 (# 3) and 1 to 329 and 676 to 1160 ( -3) of # 138, fused to Gal4 (1-147), are able to activate the transcription of the UASG-SNR6 matrix to give a level of transcripts corresponding respectively to 15% and 45% of the level of transcripts produced by the construct # 138-Ga14 (1-147) comprising the entire sequence of # 138.

 The inventors have now observed that transcription of the UASG-SNR6 gene activated by the constructs i3-Gal4 (1-147) and Tl-3-Gal4 (1-147) was sufficient to ensure cell viability.

 For this, the yMCM616 strain (MARSOLIER et al., J. Mol Biol., 1997, publication cited above), whose chromosomal SNR6 gene was inactivated by a deletion of 2 bp in its B block and which survives thanks to a copy. SNR6 gene carried by the plasmid pRS316 / SNR6 having the URA3 marker, was transformed by plasmids bearing the UASG-SNR6 construct (pRS315 / UASGSNR6) and the T3-GAL4 (1-147), il-3-GAL4 fusions. (1-147) and T138-GAL4 (1-147). The transformants were streaked on Petri dishes containing 5-FOA and incubated at 30 ° C.

<Desc / Clms Page number 25>

All transformants containing pRS315 / UASG-SNR6 and the T3-GAL4 (1-147), T1-3-GAL4 (1-147) or # 138-GAL4 (1-147) fusions were found to be able to lose the plasmid carrying the URA3 marker and wild-type copy of the SNR6 gene, and push to 30 C, demonstrating that the fusions # 3- Gal4 (1-147) and Tl-3-Gal4 (1-147) sufficiently activated the UASG-SNR6 gene to allow normal cell growth.

 Isolated transformants containing pRS315 / UASG-SNR6 and T3-GAL4 (1-147) or # 1-3-GAL4 (1-147) or # 138-GAL4 (1-147), and having lost pRS316 / SNR6, were then have been restrained on selective medium and incubated at different temperatures. Transformants containing T138-GAL4 (1-147) could grow at 34 ° C and 38 ° C, whereas transformants containing T3-GAL4 (1-147) or Tl-3-GAL4 (1-147) could grow at 34 ° C. C, but not at 38 C.

fusions i3-Gal4(1-147), ou il-3-Gal4(1-147), ou, à titre de témoin, la fusion T138-GAL4-(1-147) ont été cultivées pendant la nuit dans un milieu dépourvu de leucine et de tryptophane, diluées à une DO de 0,2 dans le même milieu et mises à pousser 8 heures supplémentaires. 100 l de culture ont alors été étalés et incubés à 38 C. Par ailleurs, les cultures ont été diluées 10 000 fois et 100 l des dilutions ont été étalés et incubés à 30 C. YMCM616 cell cultures containing the

i3-Gal4 (1-147), or il-3-Gal4 (1-147) fusions, or, as a control, the T138-GAL4- (1-147) fusion were grown overnight in a medium without leucine and tryptophan, diluted to an OD of 0.2 in the same medium and allowed to grow an additional 8 hours. 100 l of culture were then spread out and incubated at 38 ° C. In addition, the cultures were diluted 10 000 times and 100 μl dilutions were spread out and incubated at 30 ° C.

Petri dishes were examined after 4 days.

The results are shown in Table II.

~~~~~~~~~~~~~~~~~~~~~~ Table

<Tb>
<tb> Constructions <SEP> Number <SEP> of <SEP> colonies <SEP> to <SEP> 30 C <SEP> Number <SEP> of <SEP> colonies <SEP> to <SEP> 38 C
<tb># 3-GAL4- (1-147) <SEP> 83 <SEP> 1
<tb># 1-3-GAL4- (1-147) <SEP> 100 <SEP> 25
<tb> t138-GAL4- (1-147) <SEP> 110 <SEP>> 100 <SEP> 000 <SEP>
<Tb>

These results show that # 3- Gal4 (1-147), # 1-3-Ga14 (1-147) fusions are thermosensitive activators of the UASG-SNR6 gene.

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plasmide p4l6CYCl/GAL4BDP26L-Tl38 a été modifié afin d'éliminer les sites EcoRI et ClaI du MCS. Pour cela, ce plasmide a été digéré par les enzymes SalI et SmaI, et pourvu de bords francs, puis religué sur lui-même. Le plasmide modifié a ensuite été digéré par les enzymes EcoRI et Clal pour éliminer le fragment EcoRI-ClaI correspondant aux acides aminés 329 à 676 de #138. Vectors allowing the expression of a thermosensitive mutant of T138 fused to a thermosensitive mutant of Gal4BD
To construct the mutant Gal4BDP26L / # 1-3, the

Plasmid p416CYCl / GAL4BDP26L-Tl38 was modified to remove EcoRI and ClaI sites from MCS. For this, this plasmid was digested with SalI and SmaI enzymes, and provided with sharp edges, and then religated on itself. The modified plasmid was then digested with EcoRI and ClaI enzymes to remove the EcoRI-ClaI fragment corresponding to amino acids 329 to 676 of # 138.

p416CYC1/GAL4BDP26L-T1-3, ou, à titre de témoin positif, le vecteur pQBD/#138, ont été cultivées pendant la nuit dans un milieu sélectif diluées à une DO de 0,2 dans le même milieu et mises à pousser 4 heures supplémentaires. The plasmid obtained, called p416CYCl / GAL4DBP26L-T1-3, was introduced into the strain W303 / UASG-SNR6 + pQAR1 + pQBD / # 138. After removing plasmid pQBD / Tl38, the heat sensitivity of this strain was tested as follows
Cell cultures containing the vector

p416CYC1 / GAL4BDP26L-T1-3, or, as a positive control, the vector pQBD / # 138, were cultured overnight in selective medium diluted to an OD of 0.2 in the same medium and grown 4 overtime.

100 l of culture were then spread out and incubated at 38 ° C. In addition, the cultures were diluted 10 000 times and 100 μl dilutions were spread out and incubated at 30 ° C.

Petri dishes were examined after 4 days.

 The results are illustrated in Table III.

Table III

<Tb>
<tb> Constructions <SEP>, <SEP> IIJL ~ T <SEP> Number <SEP> of <SEP> colonies <SEP> to <SEP> 30 C <SEP> Number <SEP> of <SEP> colonies <SEP> to <SEP> 38 C
<Tb>

GAL4(1-147)""0L-t1-3 105 2

GAL4 (1-147) "" 0L-t1-3 105 2

<Tb>
<tb> GAL4 (1-147) - # 138 <SEP> 110 <SEP>> 100 <SEP> 000 <SEP>
<Tb>

These results demonstrate the highly heat-sensitive nature of the Ga14BDP26L- # 1-3 protein. When plasmids pQBD / # 138 or (pQBD / PRP9 + pTAU / PRP21) are introduced, the growth is restored to 38 ° C., and the strain also grows on medium containing uracil and 5-FOA.

Claims (18)

1) Chimeric transcription factor constituted by a fusion protein comprising: a domain I chosen from: the subunits of a transcription factor PolIII, and the conditional mutants of said subunits, and a domain II chosen from: the peptide domains of DNA binding and the conditional mutants of said domains characterized in that at least one of the domains I or II is a conditional mutant.  2) chimeric transcription factor according to claim 1, characterized in that the domain I is selected from subunits # 138, # 131, # 95 and T91 of the factor TFIIIC and the conditional mutants of said subunits, and the domain It is selected from the peptide-binding DNA domains of PolII transcription factors.  3) chimeric transcription factor according to any one of claims 1 or 2, characterized in that the domain I and / or the domain II is constituted by a thermosensitive mutant.  4) chimeric transcription factor according to claim 3, characterized in that its domain I is constituted by a mutant of # 138 devoid of all or part of the central domain, and comprising at least the C-terminal domain.  5) chimeric transcription factor according to any one of claims 3 or 4, characterized in that its domain II is constituted by the P26L mutant of the DNA binding domain of Gal4.  6) Nucleic acid sequence encoding a chimeric transcription factor according to any one of claims 1 to 5. <Desc / Clms Page number 28>  7) A recombinant vector comprising a nucleic acid sequence according to claim 6.  8) A yeast cell transformed with a nucleic acid sequence according to claim 6.  9) transformed yeast cell according to claim 8, wherein an X gene, transcribed by RNA polymerase III and essential to cell viability has been inactivated, and which comprises at least one copy of an X gene derived from said gene X by inactivation of an SC region of the promoter of said gene allowing the fixation of a transcription factor by polymerase III and insertion of an SC 'region allowing the attachment, under permissive conditions, of a chimeric transcription factor according to a any of claims 1 to 5.  10) yeast cell according to claim 9, characterized in that it further comprises a gene X '' derived from the X gene by inactivation of the SC region, and its replacement by a SC '' region, different from SC ', and allowing the attachment of a DNA binding peptide domain other than domain II of a chimeric transcription factor according to the invention, and other than a DNA peptide binding domain of a factor of transcription by polymerase III.  11) transformed yeast cell according to any one of claims 9 or 10, characterized in that the X 'gene is a gene derived from SNR6 by inactivation of the B block and insertion of a UASG sequence.  12) A method for detecting the interactions between two peptides or peptide domains T1 and T2, characterized in that it comprises: i) the co-transformation of at least one yeast cell according to any one of claims 9 to 11, by the sequences following DNAs: a DNA sequence (a) coding for the peptide or peptide domain T1, fused to a subunit of a transcription factor PolIII; <Desc / Clms Page number 29>  a DNA sequence (b) comprising a sequence encoding a fusion protein comprising a DNA binding peptide domain capable of binding to the SC 'region of the X' gene, and the peptide or T2 peptide domain; ii) determining the viability of said cell placed under non-permissive conditions for the chimeric transcription factor hosted by said cell.  13) Method according to claim 12, characterized in that a host cell according to claim 10 is used, and in that the sequence (b) comprises instead of the sequence encoding a peptide domain of DNA binding. capable of binding to the SC 'region of the X' gene, a sequence encoding a DNA binding peptide domain capable of binding to the SC '' region of the X gene.  14) Kit for the implementation of a method according to any one of claims 12 or 13, characterized in that it comprises: - at least one strain of transformed yeast cells according to any one of claims 9 to 11; and at least one vector comprising a DNA sequence coding for a subunit of a transcription factor PolIII and at least one site allowing insertion of a coding sequence for a polypeptide fused to said subunit, and or at least one vector comprising a DNA sequence coding for a fusion protein comprising a DNA binding peptide domain capable of binding to the SC 'region of an X' gene, and at least one site allowing the insertion of a coding sequence for a fused polypeptide into said domain, or - at least one vector comprising a DNA sequence coding for a fusion protein comprising a <Desc / Clms Page number 30>  DNA binding peptide domain capable of binding to the SC region of an X '' gene, and at least one site allowing insertion of a coding sequence for a polypeptide fused to said domain 15) Kit according to claim 14, characterized in that it further comprises a pair of positive interaction control vectors, one of which comprises a DNA sequence coding for the BD domain of a PolII transcription activator, and the other comprises a DNA sequence encoding a subunit of a transcription factor PolIII, respectively fused to sequences encoding proteins capable of interacting with one another.  16) Use of a yeast cell according to any one of claims 9 to 11 for the screening of a fusion library for the search for interaction partners with a protein.  17) Use of a kit according to any one of claims 14 or 15 for the screening of a fusion library for the search for interaction partners with a protein.  18) A method for selecting interaction partners with a peptide or a peptide domain T, characterized in that it comprises: i) co-transformation of at least one yeast cell according to any one of claims 9 to 11 by a DNA sequence (a) comprising a sequence encoding a fusion protein comprising the peptide or peptide domain T and a DNA binding domain capable of binding to the SC 'region of the X' or SC gene of the X gene and a DNA sequence (b) randomly taken from a bank of test sequences each of which encodes a fusion protein comprising a candidate Tx peptide or peptide domain. <Desc / Clms Page number 31>  the interaction with the T protein, and a Pol III transcription activator; ii) selecting viable transformed cells under non-permissive conditions for the chimeric transcription factor expressed by said cells; iii) sequencing the DNA sequence (b) recovered from the viable cells, and determining the sequence of the peptide or peptide domain Tx.